This invention relates to the diagnosis and prevention of equine diseases caused by the protozoan parasite, Neospora. The invention specifically relates to isolated cultures of the parasite and nucleic acids and proteins isolated from them.
Neospora caninum, a cyst-forming parasite, was first reported to cause paralysis in young dogs (Bjerkas, et al., Acta Pathologica Microbiologia Immunologia Scandinavica 96:445 (1984); and Dubey, et al., J. Am. Vet. Med. Ass'n 192:1269 (1988)). Naturally occurring neonatal or fetal infections caused by Neospora-like protozoa have been described in cattle, deer, goats, horses and sheep (Barr, et al., J. Vet. Diag. Invest. 3:39 (1991); Woods, et al., J. Vet. Diag. Invest. 6:508 (1994); Barr, et al., J. Vet. Diag. Invest. 4:365 (1992); Dubey, et al., J. Parasitology 78:532 (1990); Gray, et at., J. Vet. Diag. Invest. 8:130 (1996); Marsh, et al., J. Am. Vet. Med. Ass'n 209:1907 (1996); Dalt, et al., Equine Vet. J. 1997; and Dubey, J. Protozoology 2:40 (1992)).
Since the discovery of the genus Neospora, successful isolation of these parasites from dogs and cattle have been reported (Dubey, et al., J. Am. Vet. Med. Ass'n 193:1259 (1988); Conrad, et al., Parasitology 106:239 (1993); Stenlund et al., Parasitology Res. 83:214 (1997); and Yamane, et al., Res. in Vet. Sci. 63:77 (1997)), and recently a Neospora sp. isolate was isolated from a neurologically impaired horse (Marsh, et al., J. Am. Vet. Med. Ass'n 209:1907 (1996)).
Previous characterization studies have failed to identify any distinct differences between the canine and bovine isolates of Neospora caninum (Marsh, et al., J. Parasitology 81:530 (1995); Stenlund, et al., Parasitology Res. 83:214 (1997); and Yamane, et al., Res. Vet. Sci. 63:77 (1997)). Prior to the work described herein, the ecuine Neospora isolate has not been characterized or immunochemically compared to the bovine or canine isolates.
Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses. Symptoms of the disease include ataxia; weakness and spasticity, particularly of the hind legs; and difficulty controlling facial muscles. Secondary condition caused by EPM include, but are not limited to, muscle atrophy, loss of function, upward fixation of the patella, and back soreness.
Conventional thought has been that EPM is caused by Sarcocystis neurona. Sarcocystis falcatula also has been suggested as the causative agent of EPM, but from molecular data, this parasite may be the same as S. neurona. Until the elements of Koch's postulate are met, the relationship of these two parasites remains indeterminate.
Because Sarcocystis neurona does not encyst in the horse and enter a latent stage, EPM is treated as an acute infection with antiprotozoal and/or anti-inflammatory drugs, including trimethoprim plus sulfa or sulfadiazine with pyrimethamine.
Equine Neospora can only be treated during the acute stage, of the infection. Once cysts have formed in the afflicted animal, antiprotozoal drugs have no effect on the disease. Thus, Neospora infected horses must be treated early in the progression of the infection. Because the treatment of Neospora infection is different than the treatment of Sarcocystis neurona and because it is necessary to treat the afflicted animal immediately, diagnostic tools need to be developed which can distinguish the two parasites as soon as the afflicted animal shows symptoms of EPM.
To generate an assay capable of differentiating Neospora equi from Sarcocystis neurona, the more common etiologic agent of EPM, a more complete understanding of the identity and biology of Neospora-derived equine protozoal myeloencephalitis is necessary. This requires establishing continuous in vitro cultures of Neospora isolates. Such cultures would also be valuable in the development of pharmaceutical compositions for the treatment and prevention of Neospora infections. The present invention addresses these and other needs.